Diabetic hyperglycemia is known to accelerate non-enzymatic glycosylation (NEG) of many soluble and membrane proteins. This glycosylation modifies N-terminal or lysine amines and often can profoundly alter protein function. Under our current grant we have found important functional consequences of the glycosylation of three proteins: a) glycosylation of serum albumin causes its rapid ingestion by micropinocytic vesicles of endothelium; b) glycosylation of tubulin inhibits its ability to polymerize to ordered microtubules and promotes formation of insoluble tubulin aggregates; c) glycosylation of calmodulin markedly reduces its ability to promote Ca++ dependent activation of brain phosphodiesterase and Ca++ ATPase. We propose follow-up studies with albumin to evaluate the possible role of NEG in protein leakage from retinal capillaries and the evolution of retinal and glomerular microangiopathy; follow-up studies with tubulin to seek evidence for insoluble tubulin aggregates in diabetic peripheral nerve; and follow-up studies with calmodulin as one of a group of cytoskeletal-related proteins which influence many membrane functions. We propose to continue study of non-enzymatic glycosylation of a selected set of proteins; to evaluate alteration of function of these proteins; and to examine in vivo extent of glycosylation of these proteins in diabetic and normal animals. We have selected three cell types which are relevant for the histopathologic complications of diabetes (e.g., erythrocytes, platelets, and macrophages). In view of the complex metabolic and biochemical perturbations of diabetes we propose isolation of the variable of elevated glucose concentration by in vitro incubations of cells selected for study. Within these cells we focus on specific cytoskeletal proteins which are well characterized available to us in pure form, and are ideal for biochemical-functional evaluation (e.g., tubulin, actin, myosin and calmodulin). In several cases proteins appear to contain lysine residues in regions important for function, and in each case there are cogent reasons for suspecting that altered function of the particular protein may play a role in the molecular pathogenesis of diabetes (e.g., microangiopathy, neuropathy, infectious, and atheromatous diatheses). Moreover, the dysfunction of such cytoskeletal proteins, resulting from NEG, may correlate with those cellular dysfunctions which reflect elevated glucose concentration in poorly controlled diabetes.